Method for detection or measuring plasmocytoma cells

ABSTRACT

A method of detecting or determining plasmacytoma cells in a sample, said method comprising amplifying a polynucleotide that is specifically or strongly expressed in said plasmacytoma cells, and then detecting or measuring the amplified product.

TECHNICAL FIELD

The present invention relates to methods for detecting and measuringplasmacytoma cells in a sample.

BACKGROUND ART

Plasmacytoma, represented by multiple myeloma, is generally termed amyeloma, and is pathologically divided into multiple myeloma, plasmacell leukemia, solitary plasmacytoma, extramedullary plasmacytoma,smoldering multiple myeloma, or asymptomatic myeloma. All of them are Bcell tumors and are characterized by abnormal growth of monoclonalplasma cells (antibody-producing cells). Many of these tumors accumulatein the bone marrow and are diseases of poor prognosis accompanied bysystemic bone lesions.

In multiple myeloma, terminally differentiated B cells or plasma cellsthat produce and secrete immunoglobulins monoclonally increasepredominantly in the bone marrow, and thereby monoclonal immunoglobulins(M proteins) or their constituent light chains and/or H chains aredetected in the blood and urine. With the aging of the population inrecent years, patients with multiple myeloma are growing in number, andthe mortality rate has reached 2.1 in 100,000 people in Japan.

The diagnosis of multiple myeloma has been made based on the detectionof M proteins and/or bone lesions. These methods, however, required thatthe number of myeloma cells to be detected amounted to 0.2 to 1.2×10¹²,systemically, and even for the detection of M proteins, growth ofmyeloma cells to about 1.2×10¹⁰ was required. This rendered the earlydetection thereof difficult.

Furthermore, since these methods of diagnosis do not always reflect theamount of tumor cells, it was inconveniently impossible to decideeffective regimens for treatment. In addition, it was also difficult todifferentiate from benign monoclonal immunoglobulinemia in which Mproteins are similarly detected, or to diagnose the non-secretory typemyeloma in which no M proteins are present.

On the other hand, there have been attempts to diagnose myeloma usingantibodies that recognize antigen on the cell membrane of plasma cells.These antibodies include such monoclonal antibodies as anti-PCA-1(Anderson, K. C. et al., J. Immunol. (1983) 130, 1132), anti-PC-1(Anderson, K. C. et al., J. Immunol. (1983) 132, 3172), anti-MM4 (Tong,A. W. et al., Blood (1987) 69, 238), anti-CD38 (Epstein, J. et al., N.Eng. J. Med. (1990) 322, 664, Terstappen, L. W. M. M. et al., Blood(1990) 76, 1739, Leo, R. et al., Ann. Hematol. (1992) 64, 132,Shimazaki, C. et al., Am. J. Hematol. (1992) 39, 159, Hata, H. et al.,Blood (1993) 81, 3357, Harada, H. et al., Blood (1993) 81, 2658,Billadedeau, D. et al., J. Exp. Med. (1993) 178, 1023) and the like.Since such detection using antibody required a sample that contained alarge amount of myeloma cells, it was particularly unsuitable for earlydetection in which myeloma cells were not always present.

On the other hand, Goto, T. et al. reported that they immunized micewith human plasma cells and obtained a mouse monoclonal anti-HM1.24antibody that recognizes an antigen having a molecular weight of 29-33kDa specifically expressed on B cell lines (Blood (1994) 84, 1922). Itwas further reported that the antigen (HM1.24) recognized by monoclonalanti-HM1.24 antibody is considered to be an antigen associated with theterminal differentiation of B cells (Goto, T. et al., Jpn. J. Clin.Immun. (1992) 16, 688), and that monoclonal anti-HM1.24 antibody reactsto plasmacytoma in a specific manner (Shuji Ozaki et al., the program ofThe 19th General Meeting of Japan Myeloma Study Group, Generalpresentation 3). It was not known, however, that the diagnosis ofplasmacytoma could be effected using the gene of HM1.24.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method thatpermits a simple and sensitive detection or measurement even with asmall amount of tumor cells, and that permits the diagnosis ofplasmacytoma.

In order to attain the above object, the present inventors have deviseda combination of primers and a probe based on the already-known sequenceof the HM1.24 gene (Japanese Patent Application No. 9-271536), attemptedto detect and measure the amount expressed of HM1.24 mRNA in a simpleand sensitive manner and to quantitate the amount expressed. As a resultof intensive research, the present inventors have found thatplasmacytoma cells can be specifically detected or measured byPCR-amplifying HM1.24 mRNA from a small amount of tumor cells and usinga probe labeled with fluorescence, and that plasmacytoma can bediagnosed by amplifying similarly normal cells or tumor cells other thanplasmacytoma cells as a control sample and then comparing the amount ofthe amplified product, and thereby have completed the present invention.

Thus, the present invention provides a method of detecting ordetermining plasmacytoma cells in a sample, said method comprisingamplifying a polynucleotide that is specifically or strongly expressedin plasmacytoma cells and then detecting or measuring the amplifiedproduct.

As said plasmacytoma, there can be mentioned multiple myeloma, plasmacell leukemia, solitary plasmacytoma, extramedullary plasmacytoma,smoldering multiple myeloma, asymptomatic myeloma and the like.

Preferably, said polynucleotide is DNA or mRNA, in particular mRNA, thatencodes the HM1.24 antigen.

Preferably, said amplification method is the PCR method.

Preferably, said measurement method is a method that employs a labeledprobe.

Preferably, said label is a fluorophore, a radioisotope, an enzyme or acombination thereof.

The present invention also provides a method of diagnosing plasmacytomaby detecting or measuring plasmacytoma cells in a sample, said methodcomprising amplifying a polynucleotide that is specifically or stronglyexpressed in said plasmacytoma cells, detecting or measuring theamplified product, and then comparing the amount of the amplifiedproduct to that in a control sample.

EMBODIMENT FOR CARRYING OUT THE INVENTION

Plasmacytoma that is the subject of the detection or measurement methodof the present invention is a tumor that expresses the HM1.24 antigenhaving the amino acid sequence as set forth in SEQ ID NO: 1 or the aminoacid sequence substantially identical to said amino acid sequence, andincludes multiple myeloma, plasma cell leukemia, solitary plasmacytoma,extramedullary plasmacytoma, smoldering multiple myeloma, asymptomaticmyeloma and the like.

Samples containing plasmacytoma cells may be prepared by isolatingleukocytes from the peripheral blood or the bone marrow of patients withplasmacytoma and collecting RNA therefrom. Since many of the myelomastend to form a tumor mass in the bone marrow, it is desirable to collectleukocytes containing plasmacytoma cells by the bone marrow puncture tosaid tumor mass. In cases where there is a marked appearance of tumorcells in the peripheral blood as in plasma cell leukemia etc., the useof peripheral blood is preferred because of simplicity in handling.

In order to isolate leukocytes containing plasmacytoma cells from theperipheral blood or the bone marrow collected from patients withplasmacytoma, a commercially available reagent may be used forseparation by gradient centrifugation. As the commercially availablereagent, there can be used Ficoll-Paque (manufactured by PHARMACIABIOTECH), a Mono-Poly Resolving Medium (manufactured by DainipponPharmaceuticals), and the like. It is also possible to prepareleukocytes by lysing red blood cells in a hypotonic buffer and thenwashing by centrifugation. As a solution used in washing, there can beused isotonic solutions such as PBS and MEM.

In order to prepare RNA from the thus obtained leukocytes, a method wellknown to those skilled in the art may be used (see, for example, IdensiSousa Jikkenhou (Experiments on Genetic Manipulation), Yasutaka Takagi,Kodansha Scientific, pp. 24-39, 1980). RNA may be total RNA or mRNA.Total RNA or mRNA can be extracted using a commercially available kit,for example TRIZOL (manufactured by GIBCO BRL), ISOGEN (manufactured byWako Pure Chemicals Industries, Ltd.), and mRNA Purification Kit(manufactured by PHARMACIA BIOTECH).

Any target polynucleotide can be used as long as it is specifically orstrongly expressed in plasmacytoma cells. However, due to ease ofdetection, it is preferably DNA or mRNA, in particular mRNA, encodingthe HM1.24 antigen that is specifically expressed in plasmacytoma cells.

In accordance with the present invention, a specific example of astrongly expressed polynucleotide includes a polynucleotide that isexpressed five-fold or more, preferably 10-fold or more, and mostpreferably 20-fold or more than normal cells or tumor cells other thanplasmauytoma cells.

A relative amount of the polynucleotide expressed in a sample may bedetermined by subjecting RNA or mRNA extracted from said cells toagarose gel electrophoresis, allowing the product to be adsorbed to amembrane, performing Northern blot hybridization using a radio-labeledprobe, and then determining and comparing the radioactivity.Alternatively, the competitive RT-PCR method (Competitive RNATranscription Kit, Takara) may be used to amplify, at the same time, theRNA competitor and the mRNA of interest in the same reaction mixturewith the same primer. Then, based on the ratio of the amplifiedproducts, the original amount of mRNA can be estimated. Furthermore, itcan be simply determined using ABI PRISM7700 according to the methoddescribed in Examples.

As the method of amplification, the PCR method (polymerase chainreaction method) can be used. When the polynucleotide of interest ismRNA, the use of RT-PCR is preferred. For the PCR method or the RT-PCRmethod, a commercially available reagent may be used including theTaKaRa RNA LA PCR™ Kit (AMV) Ver 1.1 (manufactured by Takara Shuzo), theRT-PCR high-Plus-(manufactured by Toyoboseki), or the TaqMan EZ RT-PCRKit (manufactured by Perkin-Elmer) and the like.

As a method of amplification, in addition to the PCR method, the TMAmethod (Transcription Mediated Amplification method: Japanese PCTPublication No. 4-500759) that amplifies RNA may be used.

The primer is a pair of oligonucleotides that can amplify a segment ofthe nucleotide sequence as set forth in SEQ ID NO: 1, specifically apair of oligonucleotides that can hybridize, at a certain interval, to anucleic acid having the nucleotide sequence as set forth in SEQ ID NO: 1or a nucleic acid complementary thereto. The distance on the sequence ofSEQ ID NO: 1 to which the pair of oligonucleotides hybridize is, but isnot limited to, a range of 30 to 400 nucleotides, preferably 50 to 200nucleotides.

The primer is preferably selected from the region in which the aminoacid sequence is well conserved among the species and mainly in theextracellular region from positions 48 to 124 in the amino acid sequenceof SEQ ID NO: 1.

Based on the above conditions, the primer can be selected using, forexample, a commercially available analytic al software such as PrimerExpress (manufactured by Perkin-Elmer).

The length of the primer to be used for amplification is, but is notlimited to, 10 to 100 nucleotides, preferably 10 to 50 nucleotides, morepreferably 15 to 30 nucleotides, and more preferably 20 to 25nucleotides, for example about 20 nucleotides.

Specific examples of such a primer pair include the forward primer (SEQID NO: 3) (corresponding to positions 153 to 172 in the nucleotidesequence as set forth in SEQ ID NO: 1) and the reverse primer (SEQ IDNO: 4) (corresponding to positions 304 to 323 in the nucleotide sequenceas set forth in SEQ ID NO: 1).

The probe is an oligonucleotide that specifically hybridizes to anucleotide sequence defined by said one pair of primers. Thus, such aprobe is selected from the region between the forward primer and thereverse primer. Though the positions on SEQ ID NO: 1 corresponding tothe probe depend on the sites to which the primer pair hybridizes, thosethat hybridize to the region of nucleotide number 173 to 303 in SEQ IDNO: 1, preferably the region of nucleotide number 224 to 249 arepreferred. The length of the probe to be used is, but is not limited to,10 to 200 nucleotides, preferably 20 to 100 nucleotides, for example 25to 50 nucleotides, for example about 30 nucleotides.

The amplified polynucleotide can be detected or measured by subjectingthe PCR reaction mixture to agarose gel electrophoresis in the presenceof a luminescent substance such as ethidium bromide and then measuringthe light emitted by said band. When a probe is used, the polynucleotideis detected by allowing it to be adsorbed to a nitrocellulose membranefollowed by hybridization to the labeled probe. The sequence or thelength of the probe is not limited as long as it can specifically detectthe target polynucleotide to be detected.

For labeling, a fluorogenic substance, a radioisotope, an enzyme or acombination thereof may be used. As the fluorogenic substance, FAM(6-carboxyfluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), and HEX(6-carboxy-4,7,2′,4′,5′,7′-hexachlorofluorescein) may be used. As theradioisotope, ³²P may be used.

As the enzyme, alkaline phosphatase may be used, and as the substratefor the enzyme, CDP-Star™ (manufactured by BOEHRINGER MANNHEIM), forexample, may be used. By using ABI PRISM7700 developed by Perkin-Elmerthat permits the determination of the product with time while performingthe PCR reaction, simple determination can be attained. As the reagentfor RT-PCR in this case, TaqMan EZ RT-PCR Kit (manufactured byPerkin-Elmer) is preferably used.

EXAMPLES

The present invention will now be explained in further detail withreference to the following examples.

Example 1

Myeloma cell line U266B1 (ATCC TIB-196), KPMM2 (Deposit number P-14170,Patent application No.: Japanese Patent Application No. 6-58082), ARH-77(ATCC CRL 1621), RPMI8226 (ATCC CCL 155), and, as other tumor celllines, a T cell leukemia cell line HPB-ALL (FCCH1018) and a lung cancercell line A549 (ATCC CCL-185) were used.

Cells (5×10⁶) of each tumor cell line were centrifuged to preparepellets, which were suspended in 1 ml of TRIZOL (manufactured by GIBCOBRL) and then allowed to stand at room temperature for 5 minutes. 0.2 mlof chloroform was added thereto, which was vigorously shaken for 15seconds, allowed to stand at room temperature for 2 minutes, and thencentrifuged at 12000×g at 4° C. for 15 minutes. After centrifugation,0.5 ml of the upper layer of the solution separated into two layers wascollected and an equal amount of isopropyl alcohol was added thereto.After shaking and allowing to stand at room temperature for 10 minutes,it was centrifuged at 12000×g at 4° C. for 10 minutes. The precipitatethus obtained was washed with 1 ml of 75% ethanol and then centrifugedat 12000×g at 4° C. for 5 minutes. After drying the precipitate, it wasdissolved in 0.1 ml of a DEPC-treated water to collect RNA. The RNAconcentration was determined from the absorbance at 260nm.

The sequences of the RT-PCR primer and the probe for specificamplification and detection of HM1.24 mRNA were determined using ananalytical software, Primer Express (manufactured by Perkin-Elmer). As aresult, 5′-TCACCATCAAGGCCAACAGC-3′ (SEQ ID NO: 3) as the forward primerand 5′-AAGCCATTAGGGCCATCACA-3′ (SEQ ID NO: 4) as the reverse primer wereselected. The production of these primers was referred to AmershamPharmacia Biotech, and the product was prepared to 0.01 mM in the TEbuffer. As the probe, 5′-CATCTCCTGCAACAAGAGCTGACCGA-3′ (SEQ ID NO: 5)that bound to a fluorescent dye FAM was used and its production wasreferred to Perkin-Elmer.

Using the TaqMan EZ RT-PCR Kit (manufactured by Perkin-Elmer) as areagent for RT-PCR, RT-PCR was performed using ABI PRISM7700(manufactured by Perkin-Elmer). The PCR comprised 40 cycles. Eachspecimen was determined in triplicate wells at an amount of 25microliters per well. The solution was prepared according to theinstruction manual of the TaqMan EZ RT-PCR Kit.

Using equal amounts of RNA extracted from each cell line, RT-PCR wasperformed for 40 cycles, and the amount of fluorescence wassimultaneously determined at each step. In this case, the thresholdvalue was set at 0.05, and the number of cycle at which fluorescenceintensity exceeds 0.05 was determined for each cell. Preparing adilution series for RNA extracted from the U266B1 cell line and wasplotted with respect to the cycle number and the RNA amount, a goodstandard curve was obtained at a range of 200 to 0.32 ng. The cyclenumber for each cell was fitted to the standard curve to calculate arelative value to the amount expressed of HM1.24 mRNA for the U266B1cell line.

Thus, the amount expressed of HM1.24 mRNA for each cell was calculatedbased on the amount of HM1.24 mRNA for the U266B1 cell line, and theamount expressed of HM1.24 mRNA for the U266B1 cell line set as oneunit. In quantitation, in order to correct for variation in the amountof RNA used in RT-PCR among cell lines, the amount amplified of GAPDHmRNA was simultaneously measured using TaqMan,GAPDH Control Reagentsattached to the TaqMan EZ RT-PCR Kit, which was used for correctionassuming that an equal amount of GAPDH mRNA is present in the sample.

As a result, the amount of HM1.24 mRNA per cell was calculated asKPMM2=0.921, ARH-77=0.567, RPMI8226=0.636, U266B1=1.000, HPB-ALL=0.033and A549=0.000.

From the foregoing, little or no expression was observed in the cellsother than the multiple myeloma cells. In contrast, an expression ashigh as 0.5 to 1 unit was observed for the plasmacytoma cell lineindicating that plasmacytoma can be diagnosed using the method of thepresent invention. The result also indicates that a contamination of 10⁴myeloma cells per 10⁶ cells can be detected.

5 1 1013 DNA Homo sapiens CDS (23)..(562) DNA coding for HM1.24 antigenprotein 1 gaattcggca cgagggatct gg atg gca tct act tcg tat gac tat tgcaga 52 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg 1 5 10 gtg ccc atg gaagac ggg gat aag cgc tgt aag ctt ctg ctg ggg ata 100 Val Pro Met Glu AspGly Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile 15 20 25 gga att ctg gtg ctcctg atc atc gtg att ctg ggg gtg ccc ttg att 148 Gly Ile Leu Val Leu LeuIle Ile Val Ile Leu Gly Val Pro Leu Ile 30 35 40 atc ttc acc atc aag gccaac agc gag gcc tgc cgg gac ggc ctt cgg 196 Ile Phe Thr Ile Lys Ala AsnSer Glu Ala Cys Arg Asp Gly Leu Arg 45 50 55 gca gtg atg gag tgt cgc aatgtc acc cat ctc ctg caa caa gag ctg 244 Ala Val Met Glu Cys Arg Asn ValThr His Leu Leu Gln Gln Glu Leu 60 65 70 acc gag gcc cag aag ggc ttt caggat gtg gag gcc cag gcc gcc acc 292 Thr Glu Ala Gln Lys Gly Phe Gln AspVal Glu Ala Gln Ala Ala Thr 75 80 85 90 tgc aac cac act gtg atg gcc ctaatg gct tcc ctg gat gca gag aag 340 Cys Asn His Thr Val Met Ala Leu MetAla Ser Leu Asp Ala Glu Lys 95 100 105 gcc caa gga caa aag aaa gtg gaggag ctt gag gga gag atc act aca 388 Ala Gln Gly Gln Lys Lys Val Glu GluLeu Glu Gly Glu Ile Thr Thr 110 115 120 tta aac cat aag ctt cag gac gcgtct gca gag gtg gag cga ctg aga 436 Leu Asn His Lys Leu Gln Asp Ala SerAla Glu Val Glu Arg Leu Arg 125 130 135 aga gaa aac cag gtc tta agc gtgaga atc gcg gac aag aag tac tac 484 Arg Glu Asn Gln Val Leu Ser Val ArgIle Ala Asp Lys Lys Tyr Tyr 140 145 150 ccc agc tcc cag gac tcc agc tccgct gcg gcg ccc cag ctg ctg att 532 Pro Ser Ser Gln Asp Ser Ser Ser AlaAla Ala Pro Gln Leu Leu Ile 155 160 165 170 gtg ctg ctg ggc ctc agc gctctg ctg cag tgagatccca ggaagctggc 582 Val Leu Leu Gly Leu Ser Ala LeuLeu Gln 175 180 acatcttgga aggtccgtcc tgctcggctt ttcgcttgaa cattcccttgatctcatcag 642 ttctgagcgg gtcatggggc aacacggtta gcggggagag cacggggtagccggagaagg 702 gcctctggag caggtctgga ggggccatgg ggcagtcctg ggtctggggacacagtcggg 762 ttgacccagg gctgtctccc tccagagcct ccctccggac aatgagtcccccctcttgtc 822 tcccaccctg agattgggca tggggtgcgg tgtggggggc atgtgctgcctgttgttatg 882 2 180 PRT Homo sapiens Amino acid sequence of HM1.24antibody protein 2 Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro MetGlu Asp Gly 1 5 10 15 Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly IleLeu Val Leu Leu 20 25 30 Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile PheThr Ile Lys Ala 35 40 45 Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala ValMet Glu Cys Arg 50 55 60 Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr GluAla Gln Lys Gly 65 70 75 80 Phe Gln Asp Val Glu Ala Gln Ala Ala Thr CysAsn His Thr Val Met 85 90 95 Ala Leu Met Ala Ser Leu Asp Ala Glu Lys AlaGln Gly Gln Lys Lys 100 105 110 Val Glu Glu Leu Glu Gly Glu Ile Thr ThrLeu Asn His Lys Leu Gln 115 120 125 Asp Ala Ser Ala Glu Val Glu Arg LeuArg Arg Glu Asn Gln Val Leu 130 135 140 Ser Val Arg Ile Ala Asp Lys LysTyr Tyr Pro Ser Ser Gln Asp Ser 145 150 155 160 Ser Ser Ala Ala Ala ProGln Leu Leu Ile Val Leu Leu Gly Leu Ser 165 170 175 Ala Leu Leu Gln 1803 20 DNA Artificial Sequence Description of Artificial Sequence Forwardprimer for amplifying a part of DNA coding for HM1.24 antibody protein 3tcaccatcaa ggccaacagc 20 4 20 DNA Artificial Sequence Description ofArtificial Sequence Reverse primer for amplifying a part of HM1.24antibody protein 4 aagccattag ggccatcaca 20 5 26 DNA Artificial SequenceDescription of Artificial Sequence Probe for detecting an amplificationproduct by SEQ ID NOS 3 and 4 5 catctcctgc aacaagagct gaccga 26

What is claimed is:
 1. A method of detecting or measuring plasmacytoma cells in a sample, said method comprising amplifying DNA or mRNA that encodes the HM1.24 antigen, and then detecting or measuring the amplified product.
 2. The method according to claim 1 wherein said plasmacytoma is multiple myeloma, plasma cell leukemia, solitary plasmacytoma, extramedullary plasmacytoma, smoldering multiple myeloma, or asymptomatic myeloma.
 3. The method according to claim 1 wherein said method of amplification is the PCR method.
 4. The method according to any of claims 1, 2 or 3 wherein said method of detecting or measuring employs a labeled probe.
 5. The method according to claim 4 wherein said label is selected from the group consisting of a fluorophore, a radioisotope, an enzyme and a combination thereof.
 6. A method of diagnosing plasmacytoma by detecting or measuring plasmacytoma cells in a sample said method comprising amplifying DNA or mRNA that encodes the HM1.24 antigen, detecting or measuring the amplified product, and then comparing the amount of the amplified product to that in a control sample.
 7. The method according to claim 2 wherein said method of amplification is the PCR method.
 8. The method according to claim 2 wherein said method of detecting or measuring employs a labeled probe.
 9. The method according to claim 3 wherein said method of detecting or measuring employs a labeled probe. 